Molecular probes for multimodality imaging and tracking of stem cells

ABSTRACT

The invention relates to novel multi-modality probes for imaging, tracking and analyzing stem cells and related biological samples, and methods of preparation and use thereof. The molecular probes of the invention are constructed, for example, by utilizing (a) the high selectivity of long hydrocarbon chains for binding to plasma membranes of cells, (b) a near-infrared (NIR) dye for optical imaging, and (c) a radionuclide for PET or SPECT imaging. The in vitro and in vivo data of the optical and radiolabeled probes demonstrated their utility for detecting the presence of stem cells with multiple imaging modalities.

PRIORITY CLAIMS AND RELATED PATENT APPLICATIONS

This application claims the benefit of priority from and is the USnational phase of International Application No. PCT/US13/42786, filed onMay 27, 2013, which claims benefit of priority from U.S. ProvisionalPatent Application Ser. No. 61/654,794, filed Jun. 1, 2012, the entirecontent of each of which is incorporated herein by reference in itsentirety for all purposes.

GOVERNMENT RIGHTS

This invention was made with Government support under Grant No.1R43GM093417-01 (SBIR) awarded by the National Institutes of Health. TheUnited States Government has certain rights in the invention.

TECHNICAL FIELDS OF THE INVENTION

The invention generally relates to imaging probes. More particularly,the invention relates to novel multi-modality probes for imaging,tracking and analyzing stem cells and related biological samples, andmethods of preparation and use thereof.

BACKGROUND OF THE INVENTION

Stem cell therapies have the potential to dramatically change thetreatment of a number of diseases such as Parkinson's, Alzheimer's,spinal cord injury, diabetes, ischemia stroke and heart disease.Although tremendous achievements and rapid developments have been madein the past decade for cell-based therapies of a number of diseasestates, the potential that stem cells offer remains to be betterunderstood. One of the concerns with stem cell therapies is the riskthat transplanted stem cells could form tumors and become cancerous ifcell division continues uncontrollably. Critical insights can beachieved by observing their fate in vivo overtime using noninvasiveimaging techniques.

An emerging technology that allows for visualization of interactionsbetween molecular probes and biological targets is molecular imaging,which can be divided into two general categories: (1) the directlabeling method, and (2) the reporter gene approach. The former involvesusing an imaging-detectable probe that can be loaded into cells andwould remain intracellular during tracking. This method does not involveextensive manipulation of cells and therefore is preferred for clinicalimplementation. It has two inherent limitations: labels may be dilutedupon cell division, making the cells eventually invisible; and labelsmay efflux from cells or may degrade over time. Examples of contrastagents are FDG for PET, ¹¹¹In-oxine for SPECT and iron nanoparticles(SPIOs) for MRI, which have significant limitations. (Zhang, et al. 2008Current Pharma. Design 14 (36): 3835-3853; Zhou, et al. 2006 JACC 48(10): 2094-2106.)

The reporter gene approach is used mostly for preclinical studies.(Narsinh, et al. 2009 Molecular Imaging of Human Embryonic Stem Cells.Ch 2 p 13-32; in Methods in Molecular Biology, Viral Applications ofGreen Fluorescent Protein, Vol. 515. Barry W Hicks (Ed) Humana Press.)This approach involves inserting a reporter gene(s) into the stem cellthat can then be tracked upon administration of a reporter probe.Reporter genes are useful for assessing the longer-term survival of theimplanted cells because the reporter will be expressed as long as thecells are alive. Major disadvantages of the reporter gene approachinclude: (i) stable transfection (lentiviral or retroviral) involvesextensive molecular manipulation of the cells under study and runs therisk of insertional mutagenesis; (ii) immune reactions may be induced;(iii) uncertainties regarding the robustness of the signal remain, asthe detected signal could reflect magnitude of transgene expressioninstead of cell survival; (iv) gene modification adds additional cost;and (v) regulatory roadblocks. (Frangioni, et al. 2004 Circulation 110:3378-3384.)

To date, most stem cell tracking studies have used direct in vitro celllabeling with SPIO followed by in vivo MRI (e.g., neural stem cells weretracked in vivo for up to 18 weeks. (Guzman, et al. 2007 PNAS (USA) 104,11915-11920.) Despite the significant advantages of MRI (25-100 μmresolution, excellent anatomical and functional data), it has been foundthat iron-derived signals can persist in organs (e.g., myocardium) andcan be detected by MRI long after the cells have been destroyed, thusgenerating false-positive signals. (Wang, et al. 2008 British J.Radiology 81: 987-988; Terrovitis, et al. 2008 Circulation 117:1555-62.) SPIO labeling was also found to affect SC migration. (Schafer,et al. 2009 Cytotherapy 11 (1): 68-78.) Another significant clinicalproblem common to all MRI methods is that certain implantable devicessuch as pacemakers and defibrillators, are currently contraindicationsto MRI.

Nuclear imaging techniques, single-photon emission computed tomography(SPECT) and positron emission tomography (PET), offer high sensitivity(10⁻¹¹M-10-¹²M tracer) deep in tissue. Specialized systems of both PETand SPECT allow small animal imaging with much improved spatialresolution (1-2 mm). SPECT imaging provides 3D information and can beapplied both in small animals as well as in humans. Although thesensitivity of PET is 1-2 orders of magnitude better than SPECT, SPECTis less expensive, more widely available, and allows multispectraldetection and uses isotopes with longer half-lives. (Stodilka, et al.2006 Phys. Med. Biol. 51: 2619-2632.)

Optical imaging is a relatively new imaging modality that offersreal-time, non-radioactive, and depending on the technique,high-resolution imaging of fluorochromes embedded in diseased tissues,e.g., cellular resolution is possible using microscopy techniques. Farred (FR) and near infrared (NIR)(650-900 nm wavelengths)fluorescence-based imaging is of particular interest for noninvasive invivo imaging because of the relatively low tissue absorption, scatter,and minimal autofluorescence of FR/NIR light. The sensitivity of thismodality is comparable to nuclear techniques approaching a few thousandcells and the acquisition time is quite fast, obtaining images inseconds in most cases. (Zhang, et al. 2005 Bioconjugate Chem. 16:1232-1239.)

Current radioisotope and optical probes for stem cell tracking havelimitations. Direct cell labeling has previously been used for earlytracking of transplanted stem cells into the myocardium in clinicaltrials. The most widely used reporter gene for nuclear imaging is HSV-tkbased PET imaging using [¹⁸F]-FHBG as the reporter probe. HSV-tk has theadditional property of serving as a suicide gene upon administration ofganciclovir, thereby allowing selective ablation of stem cellmisbehavior. However, this approach suffers from many of the samelimitations previously mentioned.

Direct radiolabeling of cells has traditionally been accomplished withthe incorporation of lipophilic chelates: ¹¹¹In-oxine, ¹¹¹In-tropolone,or ^(99m)Tc-HMPAO (hexamethylpropylene amine oxime). For stem celltracking, the short radiological half-life of ^(99m)Tc (6.02 h) is notparticularly useful for longer term imaging studies using the directradiolabeling approach. MSCs and endothelial progenitor cells (EPCs)labeled with ¹¹¹In-oxine or ^(99m)Tc-HMPAO have been monitored in vivousing SPECT in animal studies (e.g., radiolabeling of progenitor cellswith ¹¹¹In is feasible for monitoring myocardial homing andbiodistribution in rats over 24-48 h. (Brenner, et al. 2004 J. Nucl.Med. 45 (3): 512-518.)

One problem reported with cells labeled with ¹¹¹In-oxine or¹¹¹In-tropolone is the potential for accumulation of the isotope in thenucleus, resulting in radiotoxicity and limiting the amount of labelpossible per cell. This is due to the short range of the emittedlow-energy B-particles, causing severe chromosomal aberration. (tenBerge, et al. 1983 J. Nucl. Med. 24: 615-620.) Evidence from a number ofstudies has shown that radiation damage from Auger-electron emitterssuch as ¹¹¹In can be reduced 85-fold if the nuclide is confined to thecytoplasm rather than the nucleus. (Lambert, et al. 1996 Nucl. Med. &Biol. 23:417-427.) If the nuclide is restricted to the cell membrane,radiation damage can be reduced 120-fold. (Hofer, K H. 1984Microdosimetry of labeled cells. In Blood Cells in Nuclear Medicine,Part II (Edited by Fueger, GF), Martinus Nijhoff Publishers, Boston;224-243.)

The cytotoxic effects of ¹¹¹In on human stem cells have also beenreported to be time-dependent. (Gholamrezanezhad, et al. 2009 Nucl. Med.Commun. 30 (3): 210-216.) Detection at the single cell level remains aformidable challenge for radionuclide probes as the ability toconcentrate radioactive agents in stem cells has yet to be achieved.(Frangioni, et al. 2004 Circulation 110: 3378-3384.)

Another important limitation that has been reported with ¹¹¹In-oxine and¹¹¹In-tropolone is the leaking of the label from the stem cell resultingin false positive signals and also high uptake in liver and kidneys.(Brenner, et al. 2004 J. Nucl. Med. 45 (3): 512-518; Zhou, et al. 2005J.Nucl. Med. 46 (5): 816-822.) This is because binding of these compoundsto intracellular structures is reversible.

SPECT has been used to track ¹¹¹In-labeled transplanted progenitor cellsin murine, porcine and canine models of myocardial infaction until 14days. (Aicher, et al. 2003 Circulation 107, 2134-9; Chin, et al. 2003Nucl. Med. Commun. 24, 1149-54; Wisenberg, et al. 2009 J. CardiovascularMagnetic Reson. 11: 11-26.) Jin et al. demonstrated that stem cells canbe radiolabeled with indium up to 0.14 Bq/cell without affectingviability and function, and detected as few as 3600 cells soradiolabeled by SPECT in a phantom study. (Jin, et al. 2005 Phys. Med.Biol. 50: 4445-4455.)

A dual modality cell labeling probe reported is ¹²⁵I-PKH95. (Slezak, etal. 1991 Nature 352:261-262; Ford, et al. 1996 J. Surgical Res. 62 (1):23-28.) The ¹²⁵I-PKH95 compound contains an iodine atom, a visiblefluorescent head group (em=570 nm) and two long alkyl tails that enableit to stably embed into cell membranes irreversibly. It was labeled byexchange with ¹²⁵I, but with low specific activity (15-40 Ci/mmol).(Gray, et al. 1991 J. Nucl. Med. 32: 1092.)

The use of an iodine isotope presents practical issues. First, exchangelabeling is not efficient in producing a high specific radioactivityagent. High specific-activity is necessary to minimize the total numberof dye molecules associated with each cell to diminish detrimentaleffects on cellular integrity. Second, it is challenging to conform to asimple kit format that can be easily radiolabeled on site.

Thus, new dual- or multi-modality probes for stem cell tracking areneeded. Labeling stem cells with new dual- or multi-modality probes andtracking them via noninvasive imaging techniques may hold the key toaddressing critical issues associated with successful development ofstem cell therapies.

SUMMARY OF THE INVENTION

The invention is based on the unexpected discovery of novel, dual- ormulti-labeled molecular probes having one or more fluorophore (e.g., afar-red fluorophore), one or more radionuclide (e.g., ¹¹¹In), and one ormore long hydrocarbon tails (e.g., two hydrocarbon tails). The probes ofthe invention incorporate into the plasma membrane of a target cellwhere incorporated probes are stable and non-diffusible. These probesallow dual- or multi-modality imaging of stem cells, providing moreaccurate determination of stem cell biodistribution in vivo.

The synergistic combination of radiologic and fluorescent labels in asingle molecular probe that can be securely attached to the cellmembrane prevents certain problems of existing probes, such as elutionof the labels from the cell. The elution issue has strained theradiologic labels for SPECT (¹¹¹In) or PET (⁶⁴Cu) alike. Since theprobes are restricted to the membrane, they exhibit minimalradiotoxicity, thereby allowing for a greater number of radiolabels percell and thus improved sensitivity.

In one aspect, the invention generally relates to a compound of theformula:

wherein

-   B is a chelating moiety capable of complexing to a radioactive    metal;-   R and R₁ are substituent groups independently selected from the    group consisting of alkyl, alkenyl, alkynyl, alkaryl and aralkyl,    each of R and R₁ comprising one or more linear or branched    hydrocarbon chains having from 2 to about 30 carbon atoms, and each    of R and R₁ being unsubstituted or substituted with one or more    non-polar functional groups;-   L comprises a fluorophore with emission in the range from about 650    nm to about 850 nm;-   R₂ is a spacer moiety having the formula:

—(R₃)_(p)-Q-(R₄-Q′)_(q)-(R₅-Q″)_(r)-(R₆-Q′″)_(s)-(R₇Q″″)_(t),

-   -   wherein

-   R₃ is an aliphatic hydrocarbon, R₄, R₅, R₆ and R₇ are independently    selected from the group consisting of aliphatic, alicyclic or    aromatic hydrocarbons, heterocycles or CH₂C(CO₂H)═CH,    -   Q, Q′, Q″, Q′″ and Q″″ are linking groups independently selected        from the group consisting of amide, thiourea, hydrazone,        acylhydrazone, ketal, acetal, orthoester, ester, anhydride,        disulfide, urea, carbamate, imine, amine, ether, carbonate,        thioether, sulfonamide, carbonyl, amidine and triazine, a        valence bond, the aliphatic or alicyclic hydrocarbons having        from about 1 to about 12 linear carbon atoms, and the aromatic        hydrocarbons having from about 6 to about 12 carbon atoms;    -   p, q, r, s and t each is 0 or 1; and

-   n is 0 or 1.

In certain preferred embodiments, B is complexed to ¹¹¹In ion.

In another aspect, the invention generally relates to a compound havingthe formula:

In yet another aspect, the invention generally relates to a compoundhaving the formula:

In yet another aspect, the invention generally relates to a method forimaging, tracking and/or analyzing cells. The method includes: labelinga sample of cells with a compound of the invention; and imaging thecells via two or more modalities comprising optical imaging andradiological imaging. In certain preferred embodiments, the cells arestem cells.

BRIEF DESCRIPT OF THE DRAWINGS

FIG. 1. Chemical structure of MTTI-157.

FIG. 2. Fluorescence microscopy of TC1 wild type mES cells labeled withMTTI-157 at 2 μM (Row 2) and 10 μM (Row 3) on Days 0, 1 and 4 afterlabeling and seeding. Unlabeled normal TC1 wild type mES cells were usedas control (Row 1). Magnification: 100×.

FIG. 3. SPECT/CT image of mouse injected with ¹¹¹In-MTTI-157 labeledstem cells by tail vein at 1 h and 19 h after injection.

FIG. 4. Mouse SPECT/CT images at 30 min and 18 h after subq injection of¹¹¹In-MTTI-157 labeled stem cells on the shoulder.

FIG. 5. Whole body count through day 9 after injection of ¹¹¹In-MTTI-157labeled stem cells by tail vain and subcutaneously on the shoulder.Corrected for decay.

FIG. 6. Optical images of a mouse through day 14 after injection with¹¹¹In-MTTI-157 labeled stem cells subcutaneously on the left shoulder.

FIG. 7. Optical images of teratoma from mouse injected subcutaneously onthe shoulder, liver, lung and spleen from mouse injected by tail vein.

FIG. 8. Fluorescence microscopy of frozen sections from liver, lung, andspleen from a mouse sacrificed on day 10 after injection by tail vein of¹¹¹In-MTTI-157 labeled stem cells, and a teratoma from mouse on day 14with subcutaneous injection on the shoulder. The first row, redfluorescence is ¹¹¹In-MTTI-157; the second row, green fluorescence isdye D275 (Ex: 484 nm, Em: 501 nm) which was used here to stain the fieldof view. Magnification: 200×.

FIG. 9. H&E staining of frozen sections from liver and lungs from mouseafter injection by tail vein of ¹¹¹In-MTTI-157 labeled stem cells. Thered arrow shows the teratoma in the sections. Magnification: 100×.

FIG. 10. Flow cytometry analysis of mES cells B/L 6 labeled withMTTI-157 conjugated with DOTA at the concentrations of 2 μM, 10 μM and20 μM.

FIG. 11. SPECT/CT imaging of mouse injected ¹¹¹In-MTTI-157 labeled stemcells by tail vein at 1 h, 22 h and day 4 after injection.

FIG. 12. SPECT/CT images of a mouse injected ¹¹¹In-MTTI-157 labeled stemcells subcutaneously on the left shoulder. Views are from 30 min, 21 hand day 4 after injection of cells.

FIG. 13. Optical images of a mouse injected with ¹¹¹In-MTTI-157 labeledstem cells subcutaneously on the shoulder through. Images shown arelateral views and were taken through day 11. Red arrow indicates site ofcell injection site.

FIG. 14. Optical images of the teratoma from the mouse injectedsubcutaneously on the shoulder, and the liver, lung and spleen from themouse injected by tail vein. Red circle shows the muscle in the fieldfor background signal.

FIG. 15. Fluorescence microscopy of frozen sections from liver, lung,and spleen from mouse with iv tail injection. The teratoma is from thesubcutaneous injection. The top row, red fluorescence is ¹¹¹In-MTTI-157;the bottom row, green fluorescence is dye D275 (Ex: 484 nm, Em: 501 nm)which stained the cell membrane and was used here to stain the field ofview. Magnification: 100×.

FIG. 16. H&E staining of frozen sections from liver and lungs. The redarrow indicates the teratoma within the tissue sections. Magnification:100×.

FIG. 17. C8 HPLC of ¹¹¹In-MTTI-157 the product eluted at 18 min as onemajor peak. The labeling efficiency was greater than 90%.

FIG. 18. The results of bright light and fluorescence microscopy ofcells labeled with 20 μM ¹¹¹In-MTTI-157 are shown for days 0, 3 and 8.

FIG. 19. The radioactivity associated with cells incubated with 2, 10and 20 μm ¹¹¹In-MTTI-157 for days 0, 3, 8, and cell growth over 8 days.

FIG. 20. ¹¹¹In -MTTI-157 labeled cells (12-14 μCi on 2×10⁶ cells) wereinjected IV into SKH-1 mice. Radioactivity was observed by SPECT/CTthrough day 7 in (a) and through day 5 (b). Activity is in the lungs inthe early images and then distributes to the liver and spleen.

FIG. 21. Whole body ¹¹¹In activity through 14 days (corrected fordecay).

FIG. 22. Fluorescent images of SKH-1 mice (untreated, left panel); andfollowing injection of ¹¹¹In-MTTI-157 labeled cells at 5 min (day 0),and at intervals through day 28.

FIG. 23. Optical scans of the excised lungs, liver and spleen from miceon day 0 through day 28.

FIG. 24. Examination of lungs, liver and spleen by fluorescencemicroscopy confirm the retention of the MTTI-157.

FIG. 25. Mean fluorescence intensity per cell for dyes MT279, MT280,MT284, MT285, CN1012, and MTTI-157 at different concentration.

FIG. 26. Percent of cells labeled at four dye concentrations for dyesMT279, MT280, MT284, MT285, CN1012, and MTTI-157.

FIG. 27. Flow cytometry analysis of LS174T cells labeled with six dyesrespectively and co-cultured with unlabeled cells. Percent of totalcells labeled.

FIG. 28. Flow cytometry analysis of LS174T cells labeled with six dyesrespectively and co-cultured with unlabeled cells. Percent of totalcells labeled.

FIG. 29. Flow cytometry analysis of LS174T cells labeled with six dyesrespectively and co-cultured with unlabeled cells. Percent of totalcells labeled.

FIG. 30. Flow cytometry analysis of LS174T cells labeled with three dyesrespectively and co-cultured with unlabeled cells. Percent of totalcells labeled.

DEFINITIONS

Definitions of specific functional groups and chemical terms aredescribed in more detail below. General principles of organic chemistry,as well as specific functional moieties and reactivity, are described in“Organic Chemistry”, Thomas Sorrell, University Science Books,Sausalito: 2006. It will be appreciated that the compounds, as describedherein, may be substituted with any number of substituents or functionalmoieties.

As used herein, “C_(x)-C_(y)” refers in general to groups that have fromx to y (inclusive) carbon atoms. Therefore, for example, C₁-C₆ refers togroups that have 1, 2, 3, 4, 5, or 6 carbon atoms, which encompassC₁-C₂, C₁-C₃, C₁-C₄, C₁-C₅, C₂-C₃, C₂-C₄, C₂-C₅, C₂-C₆, and all likecombinations. C₁-C₂₀ and the likes similarly encompass the variouscombinations between 1 and 20 (inclusive) carbon atoms, such as C₁-C₆,C₁-C₁₂ and C₃-C₁₂.

As used herein, the term “C_(x)-C_(y) alkyl” refers to a saturatedlinear or branched free radical consisting essentially of x to y carbonatoms, wherein x is an integer from 1 to about 10 and y is an integerfrom about 2 to about 20. Exemplary C_(x)-C_(y) alkyl groups includeC₁-C₂₀ alkyl,” which refers to a saturated linear or branched freeradical consisting essentially of 1 to 20 carbon atoms and acorresponding number of hydrogen atoms. Exemplary C₁-C₂₀ alkyl groupsinclude methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,dodecanyl, etc. Of course, other C₁-C₂₀ alkyl groups will be readilyapparent to those of skill in the art given the benefit of the presentdisclosure.

As used herein, the term, “C_(x)-C_(y) alkoxy” refers to a straight orbranched chain alkyl group consisting essentially of from x to y carbonatoms that is attached to the main structure via an oxygen atom, whereinx is an integer from 1 to about 10 and y is an integer from about 2 toabout 20. For example, C₁-C₂₀ alkoxy refers to a straight or branchedchain alkyl group having 1-20 carbon atoms that is attached to the mainstructure via an oxygen atom, thus having the general formula alkyl-O—,such as, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy,sec-butoxy, tert-butoxy, pentoxy, 2-pentyl, isopentoxy, neopentoxy,hexoxy, 2-hexoxy, 3-hexoxy, and 3-methylpentoxy.

As used herein, the term “acyl” refers to a group or radical —C(O)R′,where R′ is hydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl,arylalkyl, heteroalkyl, heteroaryl, heteroarylalkyl as defined herein.Representative examples include, but are not limited to, formyl, acetyl,cylcohexylcarbonyl, cyclohexylmethylcarbonyl, benzoyl, benzylcarbonyland the like.

As used herein, the term “alkenyl” refers to monovalent olefinicallyunsaturated hydrocarbyl groups preferably having 2 to 11 carbon atoms,particularly, from 2 to 8 carbon atoms, and more particularly, from 2 to6 carbon atoms, which can be straight-chained or branched and having atleast 1 and particularly from 1 to 2 sites of olefinic unsaturation.Particular alkenyl groups include ethenyl, n-propenyl, isopropenyl,vinyl and substituted vinyl, and the like.

As used herein, the term “alkynyl” refers to acetylenically oralkynically unsaturated hydrocarbyl groups particularly having 2 to 11carbon atoms and more particularly 2 to 6 carbon atoms which can bestraight-chained or branched and having at least 1 and particularly from1 to 2 sites of alkynyl unsaturation. Particular non-limiting examplesof alkynyl groups include acetylenic, ethynyl, propargyl, and the like.

As used herein, the term “alkaryl” refers to an aryl group substitutedwith one or more alkyl groups.

As used herein, the term “aralkyl” refers to an alkyl group substitutedwith one or more aryl groups.

As used herein, the term “aryl” refers to a monovalent aromatichydrocarbon group derived by the removal of one hydrogen atom from asingle carbon atom of a parent aromatic ring system. Typical aryl groupsinclude, but are not limited to, groups derived from aceanthrylene,acenaphthylene, acephenanthrylene, anthracene, azulene, benzene,chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene,hexylene, as-indacene, s-indacene, indane, indene, naphthalene,octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene,pentalene, pentaphene, perylene, phenalene, phenanthrene, picene,pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthaleneand the like. Particularly, an aryl group comprises from 6 to 14 carbonatoms.

As used herein, the term “halogen” refers to fluorine (F), chlorine(Cl), bromine (Br), or iodine (I).

DETAILED DESCRIPTION OF THE INVENTION

The invention generally relates to novel dual- or multi-modality probesfor imaging, tracking and analyzing cells (e.g., stem cells) and relatedbiological samples, and methods of preparation and use thereof. Inparticular, the molecular probes of the invention are constructed byutilizing (1) long hydrocarbon chains highly-selective for binding toplasma membranes of cells, (2) a near-infrared (NIR) dye for opticalimaging, and (3) a radionuclide for PET or SPECT imaging. The in vitroand in vivo data of the optical and radiolabeled probes demonstratedtheir utility for detecting the presence of stem cells with multipleimaging modalities.

Stem cells are biological cells found in all multicellular organismsthat can divide and differentiate into diverse specialized cell typesand can self-renew to produce more stem cells. They are precursor cellsthat possess the capability for proliferation and self-renewal, and theability to regenerate into multiple cell lines. Progenitor cells referto immature or undifferentiated cells, typically found in post-nataltissues. Multiple potential sources for clinically useful stem andprogenitor cells have been identified, including autologous andallogeneic embryonic cells and fetal and adult somatic cells fromneural, adipose, and mesenchymal tissues. Mesenchymal stem cells (MSCs)harvested from bone marrow are easy to obtain and highly proliferative,allowing autologous transplantation without any need forimmune-suppression. (Herzog, et al. 2003 Blood 102: 3483-3493; Jiang, etal. 2002 Nature 418: 41-49.) In contrast to embryo-derived stem cells,MSCs pose few ethical problems.

The development of stem cell therapies requires the ability to trackcells noninvasively in vivo with agents that distribute to theirdaughter cells during division. For transplanted stem cells to engraftsuccessfully cells must: (i) survive after transplant; (ii) home to therequired site, (iii) differentiate into the required cell type; (iv)integrate into the desired tissue; and (v) function as the desiredtissue. Alternatively, they may secrete cytokines (e.g., growth factors)that promote the growth of existing cells (i.e paracrine effect).

At present, no single imaging modality possesses all the desiredqualities for optimal evaluation of stem cell therapies: (i) elucidatethe optimal stem cell type and number, (ii) develop adequateadministration methods, (iii) assess effects of delivery of biofactorson stem cell distribution and, (iv) evaluate stem cells as a componentof tissue engineered constructs.

The novel, dual- or multi-labeled molecular probes disclosed herein haveone or more fluorophore (e.g., a far-red fluorophore), one or moreradionuclide (e.g., a ¹¹¹In), and one or more long hydrocarbon tails(e.g., two tails). They incorporate into the plasma membrane of a celland the incorporated probes are stable and non-diffusible.

Thus, as disclosed herein, radiologic and fluorescent labels aresynergistically combined in a single molecular probe that can besecurely attached to the cell membrane, providing the benefits of highsensitivity and precise anatomical localization. Since the probes arerestricted to the membrane, they exhibit minimal radiotoxicity, therebyallowing for a greater number of radiolabels per cell and thus improvedsensitivity. Since the probes of the invention are designed not to“leak” from the labeled stem cell they have improved accuracy indetermination of stem cell biodistributions compared with ¹¹¹In-oxineover a period of at least 10 days. ¹¹¹In labels offer the opportunity tomonitor the cells in vivo for a term of about two weeks whereas opticalimaging using a membrane embedded FR/NIR fluorochrome offers thepotential for possibly monitoring up to at least 16 weeks. Radiologicallabels allow an evaluation of sites at depth, while optical labels areuseful for cells near the surface and for final single cell detection ofstem cells via microscopy.

In one aspect, the invention generally relates to a compound of theformula:

wherein

-   B is a chelating moiety capable of complexing to a radioactive    metal;-   R and R₁ are substituent groups independently selected from the    group consisting of alkyl, alkenyl, alkynyl, alkaryl and aralkyl,    each of R and R₁ comprising one or more linear or branched    hydrocarbon chains having from about 2 to about 30 carbon atoms, and    each of R and R₁ being unsubstituted or substituted with one or more    non-polar functional groups;-   L comprises a fluorophore with emission in the range from about 650    nm to about 850 nm;-   R₂ is a spacer moiety having the formula:

—(R₃)_(p)-Q-(R₄-Q′)_(q)-(R₅-Q″)_(r)-(R₆-Q′″)_(s)-(R₇Q″″)_(t),

-   -   wherein    -   R₃ is an aliphatic hydrocarbon, R₄, R₅, R₆ and R₇ are        independently selected from the group consisting of aliphatic,        alicyclic or aromatic hydrocarbons, heterocycles or        CH₂C(CO₂H)═CH,    -   Q, Q′, Q″, Q′″ and Q″″ are linking groups independently selected        from the group consisting of amide, thiourea, hydrazone,        acylhydrazone, ketal, acetal, orthoester, ester, anhydride,        disulfide, urea, carbamate, imine, amine, ether, carbonate,        thioether, sulfonamide, carbonyl, amidine and triazine, a        valence bond, the aliphatic or alicyclic hydrocarbons having        from about 1 to about 12 linear carbon atoms, and the aromatic        hydrocarbons having from about 6 to about 12 carbon atoms;    -   p, q, r, s and t each is 0 or 1; and

-   n is 0 or 1.

In certain preferred embodiments, B is complexed to ¹¹¹In ion.

R and R₁ may have from about 2 to about 30 carbon atoms (e.g., fromabout 5 to about 30, from about 10 to about 30, from about 15 to about30, from about 18 to about 30, from about 6 to about 25, from about 6 toabout 25).

In certain preferred embodiments, the compound has the formula:

wherein

-   Z is a substituent selected from the group consisting of H, alkyl,    OH, O-alkyl, COOH, CONH₂, SO₃H, SO₂NH₂, CONH-alkyl, CON(alkyl)₂,    NH-acyl, NH-alkyl, N(alkyl)₂, SH, S-alkyl, NO₂, halogen, Si(alkyl)₃    and O—Si(alkyl)₃, wherein the alkyl groups independently comprising    from 1 to 4 carbon atoms;-   y is 2 or 3; and-   A is a biologically compatible counter anion.

In certain preferred embodiments, B is complexed to ¹¹¹In ion.

In certain preferred embodiments, the compound has the formula:

wherein

-   Z is a substituent selected from the group consisting of H, alkyl,    OH, O-alkyl, COOH, CONH₂, SO₃H, SO₂NH₂, CONH-alkyl, CON(alkyl)₂,    NH-acyl, NH-alkyl, N(alkyl)₂, SH, S-alkyl, NO₂, halogen, Si(alkyl)₃    and O—Si(alkyl)₃, wherein the alkyl groups independently comprising    from about 1 to about 4 carbon atoms;-   X is a substituent selected from the group consisting of H, halogen,    phenoxy, thiophenoxy and aryl; and-   A is a biologically compatible counter anion.

In certain preferred embodiments, B is complexed to ¹¹¹In ion.

In certain preferred embodiments, the compound has the formula:

wherein

-   Z is a substituent selected from the group consisting of H, alkyl,    OH, O-alkyl, COOH, CONH₂, SO₃H, SO₂NH₂, CONH-alkyl, CON(alkyl)₂,    NH-acyl, NH-alkyl, N(alkyl)₂, SH, S-alkyl, NO₂, halogen, Si(alkyl)₃    and O—Si(alkyl)₃, wherein the alkyl groups independently comprising    from about 1 to about 4 carbon atoms.

In certain preferred embodiments, B is complexed to ¹¹¹In ion.

In certain preferred embodiments, the compound has the formula:

wherein

-   Z is a substituent selected from the group consisting of H, alkyl,    OH, O-alkyl, COOH, CONH₂, SO₃H, SO₂NH₂, CONH-alkyl, CON(alkyl)₂,    NH-acyl, NH-alkyl, N(alkyl)₂, SH, S-alkyl, NO₂, halogen, Si(alkyl)₃    and O—Si(alkyl)₃, wherein the alkyl groups independently comprising    from about 1 to about 4 carbon atoms.

In certain preferred embodiments, B is complexed to ¹¹¹In ion.

In certain preferred embodiments, the compound has the formula:

wherein

-   Z is a substituent selected from the group consisting of H, alkyl,    OH, O-alkyl, COOH, CONH₂, SO₃H, SO₂NH₂, CONH-alkyl, CON(alkyl)₂,    NH-acyl, NH-alkyl, N(alkyl)₂, SH, S-alkyl, NO₂, halogen, Si(alkyl)₃    and O—Si(alkyl)₃, wherein the alkyl groups independently comprising    from about 1 to about 4 carbon atoms; and-   X is a substituent selected from the group consisting of H, halogen,    phenoxy, thiophenoxy and aryl.

In certain preferred embodiments, B is complexed to ¹¹¹In ion.

In certain preferred embodiments, the compound has the formula

wherein

-   Z is a substituent selected from the group consisting of H, alkyl,    OH, O-alkyl, COOH, CONH₂, SO₃H, SO₂NH₂, CONH-alkyl, CON(alkyl)₂,    NH-acyl, NH-alkyl, N(alkyl)₂, SH, S-alkyl, NO₂, halogen, Si(alkyl)₃    and O—Si(alkyl)₃, wherein the alkyl groups independently comprising    from about 1 to about 4 carbon atoms.

In certain preferred embodiments, B is complexed to ¹¹¹In ion.

In another aspect, the invention generally relates to a compound havingthe formula:

In yet another aspect, the invention generally relates to a compoundhaving the formula:

In yet another aspect, the invention generally relates to a method forimaging, tracking and/or analyzing cells. The method includes: labelinga sample of cells with a compound of the invention; and imaging thecells via two or more modalities comprising optical imaging andradiological imaging. In certain preferred embodiments, the cells arestem cells.

Dual- or multi-modality labeling probes disclosed herein also presentsignificant commercial potential. These compounds are compatible withnumerous types of instrumentation to allow wide use and quick adoption.Clinical and small animal PET and SPECT systems are widely available asare numerous small animal optical imaging systems based uponfluorescence. For example, a patient for stem cell therapy may beadministered stem cells labeled with the dual imaging probe. Initially,the localization, quantification and clearance of the stem cells arevisualized by whole-body nuclear imaging (SPECT/CT). At later timepoints, if necessary inter operative optical probes could be used toestablish retention and differentiation at the disease site. Thus, theinvention allows optimized preparation and labeling of stem cells exvivo and tracking the cells in vivo once re-injected back into thepatient such that there are no or minimal effects on cell viability,membrane integrity, proliferation, differentiation and function. Theprobes of the invention can be used for tracking stem cells in vivo aswell as for use in cell based therapies.

Disclosed herein are various examples that demonstrate incorporation andstability of dye, viability of labeled cells, and ability of dye to becarried through to the subsequent generations of cells.

EXAMPLES

The DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid)conjugated DiD fluorescent analog (fluorescence excitation 648 nm,emission 667 nm) (MTTI-157) is shown in FIG. 1).

Example 1: Preparation of Exemplary Compounds (as Shown in Scheme 1)Preparation of Compound (1)

Compound (1) was prepared according to the procedure of Gale et al.(1977).

Preparation of Compound (2)

Compound (1) (20.6 g, 0.065 mol) andn-docosanyl-4-chlorobenzenesulfonate (32.4 g, 0.065 mol) were heatedtogether at 130° C. with stirring for 2 h. The mixture was then cooledto RT and the crude solid recrystallized from ethyl acetate to yieldpure (2) (36.4 g, 69%). ¹H NMR (CDCl₃, 200 MHz): δ7.50-7.90 (m, 8H),7.20-7.30 (m, 3H), 4.95 (s, 2H), 4.65 (t, 2H), 2.95 (s, 3H), 1.7-1.9(m), 1.55 (s, 6H), 1.10-1.40 (m), 0.85 (t, 3H).

Preparation of compound (3)

Compound (2) (36 g, 0.044 mol) was dissolved in concentratedhydrochloric acid (660 mL) and the solution slowly heated to 115° C.over 2 h and then refluxed at this temperature for 22 h. The solutionwas then cooled to RT and placed in and ice bath and then taken to pH=9with 30% ammonium hydroxide solution. The solution was diluted furtherwith water and then extracted with dichloromethane (3×300 mL). Theorganic extracts were dried over magnesium sulfate, filtered andconcentrated to provide the product (12.8 g, 57%). ¹H NMR (CDCl₃, 200MHz): δ 7.0-7.10 (m, 2H), 6.50 (d, 1H), 3.75-3.90 (m, 4H), 3.45 (t, 2H),1.90 (s), 1.50-1.70 (m), 1.10-1.40 (m), 0.85 (t, 3H).

Preparation of Compound (4)

Compound (3) (12.8 g, 0.0258 mol) was dissolved in methyl formate (50mL) and heated at reflux under an argon atmosphere for 24 h. The excessmethyl formate was then removed by rotary evaporation and the cruderecrystallized from hexane to provide pure material (7.62 g, 56%).

Preparation of Compound (5)

2,3,3-trimethyl-(3H)-indoleine (626 g, 0.04 mol) andn-docosanyl-4-chlorobenzene sulfonate (20.02 g, 0.04 mol) [Sondermann,1971] were heated together at 140° C. with stirring for 3 h. Thereaction was then cooled provide a waxy solid. The solid was dissolvedin ethanol (250 mL) and 200 mL of saturated potassium iodide was addedfollowed by stirring for 30 min. 1 L of cold water was added andstirring continued for another 15 min. The precipitate was collected byfiltration, washed with water, dried under high vacuum andrecrystallized from methylene chloride/hexane to furnish pure (5) (14.5g, 61%), mp=107-110° C. ¹H NMR (CDCl₃, 200 MHz): δ 7.50-7.80 (m, 4H),4.65 (t, J=8 Hz, 2H), 3.10 (s, 3H), 1.80-2.00 (m, 2H), 1.65 (s, 6H),1.10-1.50 (m), 0.85 (t, J=7 Hz, 3H).

Preparation of Compound (6)

A mixture of compound (5) (1 g, 1.68 mmol) and malonaldehydebis(phenylimine)monohydrochloride (0.478 mg, 1.85 mmol) in aceticanhydride (40 mL) was heated at 120° C. in an oil bath for 1 h. Aftercooling to RT, it was added to 100 mL of 10% potasssium iodide solutionand refrigerated. The solid preipitate was collected by filtration,washed with water and dried in a vacuum oven to furnish 1.5 grams ofproduct (100% yield) which was used without further purification.

Preparation of Compound (7)

Compound (6) (1.5 g, 2 mmol) and compound (4) (1.05 g, 2 mmol) wererefluxed together in dichloromethane (40 mL) for 2 h. Afterconcentration the crude material was purified by silica gelchromatography eluting with an increasing gradient of methanol (0 to 5%)in dichloromethane. The product was further purified byrecrystallization from ethanol to provide pure (7) (240 mg, 9.4%). ¹HNMR (CDCl₃, 400 MHz): δ8.35 (s, 1H), 8.09-7.98 (m, 2H), 7.75-7.68 (m,1H), 7.60 (s, 1H), 7.39 (q, 3H, J=16.3), 7.24 (t, 1H, J=7.4), 7.03 (dd,2H, J=13.6), 6.64 (t, 1H, J=12.4), 6.16 (m, 2H), 4.55 (d, 2H, J=6.2),4.00 (q, 4H, J=15.7), 1.50-1.21 (m, 68H), 0.883 (t, 6H, J=7.0). ExpectedM⁺(C₇₁H₁₁₈N₃O)=1028.93; Observed M⁺=1029.0.

Scheme 1 is a synthetic scheme for preparing non-radioactive andradioactive indium labeled compounds of the invention with a DOTAchelator. Reagents: (i) n-docosanyl-4-chlorobenzene-sulfonate, KI (ii)cHCl, heat (iii) methylformate, heat (iv) malonaldehydebis-(phenyl-imine)mono-hydrochloride, Ac₂O, heat (v) heat (vi) 47% HI(vii) DOTA-mono-NHS-tris(^(t)butylester).HPF₆, DIPEA (viii) TFA/CH₂Cl₂(60:40) (ix) EtOH, ^(nat)InCl₃, 0.25M NaOAc or EtOH, ¹¹¹InCl₃, 0.25MNaOAc.

Preparation of Compound (8)

Compound (7) (280 mg) was dissolved in 10 mL Methanol and 1 mL of 47% HIwas added and then mixture heated at 65° C. for 3 h. The reaction wasthen cooled to (0-5) ° C. in an ice bath for 1 h and the resultingprecipiated collected by filtration and washed with a small volume ofcold methanol. The product was then dried overnight in the vacuum ovenat 40° C. to provide pure (8) (255 mg, 91%). ¹H NMR (CDCl₃, 400 MHz):δ8.10 (t, 3H, J=13.0), 7.72 (d, 1H, J=7.9), 7.42 (d, 1H, J=7.3), 7.36(t, 1H, J=7.7), 7.23 (t, 1H, J=7.6), 7.10 (d, 1H, J=8.3), 7.05 (d, 1H,J=8.0), 6.64 (t, 1H, J=12.5), 6.15 (d, 2H, J=13.5), 4.36 (s, 2H),3.89-4.10 (m, 4H), 1.69-1.91 (m, 12H), 1.13-1.49 (m, 83H), 0.87 (t, 6H,J=6.6). Expected M⁺(C₇₀H₁₁₉N₃)=1000.93; Observed M⁺=1001.0.

Preparation of Compound (9)

Compound (8) (154 mg, 0.123 mmol),DOTA-mono-NHS-tris(^(t)butylester).HPF₆(Macrocyclics, TX) (100 mg, 0.123mmol), diisopropylethylamine (20 μL) were stirred in dichloromethane (5mL) at RT for 2 h. The product was then purified via silica gelchromatography eluting with dichloromethane plus an increaing amount ofmethanol (0 to 3%) to furnish pure (9) (70 mg, 28%). ¹H NMR (CDCl₃, 400MHz): δ8.04-7.93 (m, 2H), 7.49-7.43 (m, 1H), 7.37 (dd, 3H, J=16.1), 7.22(t, 1H, J=7.4), 7.09-7.00 (m, 2H), 6.53 (t, 1H, J=12.5), 6.13-5.98 (m,2H), 4.59-4.35 (m, 2H), 4.02-3.82 (m, 4H), 3.62-3.37 (m, 4H), 3.25-3.11(m, 2H), 3.06-2.77 (m, 6H), 2.25-2.06 (m, 2H), 1.83-1.70 (m, 14H),1.70-1.54 (m, 6H), 1.52-1.40 (m, 27H), 1.39-1.36 (m, 3H), 1.35-1.19 (m,68H), 0.884 (t, 6H, J=6.8). Expected M+H (C₉₈H₁₆₉N₇O₇)=1556.3; ObservedM+H=1556.0.

Preparation of Compound (10)

Compound (9) (70 mg, 0.042 mmol) was stirred in a mixture ofTFA/dichloromethane (60:40) (10 mL) at RT overnight. The reactionmixture was then concentrated and dried under high vacuum to provide theproduct (40 mg, 57% yield). ¹H NMR (DMSO-d₆, 400 MHz): δ8.79-8.66 (m,1H), 8.33-8.24 (m, 2H), 7.58 (d, 1H J=7.0), 7.47 (s, 1H), 7.36 (d, 2H,J=4.2), 7.30-7.26 (m, 1H), 7.60-7.56 (m, 1H), 6.87-6.81 (m, 1H), 6.52(t, 1H, J=12.3), 6.24 (dd, 1H, J=6.7), 4.36-4.30 (m, 1H), 4.11-3.99 (m,3H), 3.61-3.56 (m, 3H), 3.03-2.93 (m, 10H), 1.87 (s, 1H), 1.66-1.61 (m,12H), 1.37-1.08 (m, 79H), 0.803 (t, 6H, J=6.8). ExpectedM⁺(C₈₆H₁₄₄N₇O₇)=1387.0; Observed M⁺=1387.0.

Preparation of Compound (11)

Compound (8) (20 mg) was dissolved in methanol (2 mL) and a solution ofindium trichloride hexahydrate (9.9 mg) in 0.25M sodium acetate, pH=6(0.67 mL) was added. The resulting reaction mixture was stirred at RTfor 2 h (a small precipitation was observed almost immediately). Theproduct was purified via normal phase silica gel chromatography elutingwith an increasing amount of methanol (1 to 10%) in dichloromethane (8mg, 37%). Expected M⁺(C₈₆H₁₄₁N₇O₇In)=1498.8; Observed M⁺=1499.0.

Example 2: Preparation of Example Compounds (as Shown in Scheme 2)

The amine functionalized dye (8) was reacted with glutaric anhydride toprovide (12). Standard coupling of (12) with 2-(4-aminobenzyl)diethylenetriamine penta-t-butyl acetate (www.macrocyclics.com) usingHATU provided (13) which was purified via column chromatography andisolated in high yield. Removal of the t-butyl esters from (13) wasaccomplished by treating with TFA in dichloromethane to furnish the freeacid (14). (14) was used to prepare both natural and indium-111 labeled(15) using methods similar to those described above for labeling (10).

Scheme 2 shows synthetic scheme for preparing non-radioactive andradioactive indium labeled compounds of the invention with a DTPAchelator. Reagents: (i) glutaric anhydride (ii) 2-(4-aminobenzyl)diethylenetriamine penta-t-butyl acetate, HATU (iii) TFA/CH₂Cl₂ (60:40)(iv) EtOH, ^(nat)InCl₃, 0.25M NaOAc or EtOH, ¹¹¹InCl₃, 0.25M NaOAc.

Example 3. Stem Cells Labeling with MTTI-157 (No ¹¹¹In)

TC1 wild type mouse embryonic stem (mES) cells were cultured in M15medium (high glucose DMEM with 2 mM glutamine, 100 U/ml Pen/Strep, 0.1mM beta mercaptoethanol, 15% FBS and 1000 U/ml murine leukemiainhibitory factor) on a freshly prepared gelatinized 100-mm tissueculture dish at 37° C. in a humidified atmosphere with 5% CO₂. Mediumwas changed every day and cells passed every other day. For labelingwith MTTI-157, the TC1 wild type mES cells were trypsinized, collectedfrom the dishes, and washed twice with high glucose DMEM without serum.The washed cells were resuspended in Diluent C at the concentration of2×10⁷ cells/ml. Immediately, prior to staining, a 2×working stocksolution of MTTI-157 (4 μM, and 20 μM) was prepared in Diluent C. Then 1ml of the cell suspension was added rapidly into 1 ml of the 2× workingdye solution and immediately mixed by pipetting. The final stainingsolutions were 2 μM and 10 μM. After incubation for 5 min at RT withconstant mixing by pipetting up and down, the staining was stopped byadding an equal volume of 1% BSA and incubating for 1 min at RT.Subsequently, the cell suspension was centrifuged at 400 g for 10 min,and the cells were washed 3 times with 10 ml of DMEM with serum toremove unbound dye. After labeling, the cell viability was determined bytrypan blue.

The MTTI-157 labeled mES cells were seeded into 12-well plates withabout 0.5×10⁶ cells per well to grow under normal culture conditions toevaluate cell viability, differentiation, fluorescence dye stability,and dye partitioning to daughter cells over time. Unlabeled normal TC1wild type mES cells were used as control. The cell associatedfluorescence was detected from the plates on an Olympus IX 70 InvertedLight Microscope (Olympus America, Inc., NY) using a Cy5 filter on Days0, 1 and 4 after seeding.

After dye labeling, the trypan blue staining was performed to detectcell viability. The trypan blue staining analysis indicated that cellviability was more than 90% at both 2 μM and 10 μM of dye. Thefluorescence microscopy analysis on Days 0, 1 and 4 are presented inFIG. 2. The fluorescence is shown in red and paired with thephase-contrast image on its right. Row 1 (top row) shows the controlswith the unlabeled mES cells over the 4 days. The cells exhibit normalgrowth and differentiation, beginning as single cells (Day 0) andgrowing into larger multi-cellular masses over the four days. Row 2(middle row) shows the mES cells labeled with MTTI-157 at 2 μM, and Row3 (bottom row) are the cells labeled at 10 μM. It is obvious from Rows 2and 3 that the MTTI-157 incorporated into the mES cells. On Day 0 thephase-contrast image shows cells shortly after plating in their singlecellular form. By the next day (Day 1), the cells begin to merge formingclones, as expected, and the fluorescence is seen distributed within theclones with passage to other members of the cell cluster, i.e., thedaughter cells. On Day 4, the cell clusters are now larger and morecells within the cluster appear to carry the fluorescence signal.Through Day 4 cell viability was preserved at both 2 μM and 10 μM dyewith no obvious effects on cellular differentiation.

Cell viability and differentiation not effected by dye concentration.

Example 4. SPECT/CT and Optical Imaging of TC1 Wild Type Mouse EmbryonicStem Cells Labeled with ¹¹¹In-MTTI-157 in Nude Mice: First Example

All animal experiments were approved by the Institutional Animal Careand Use Committee of the university. Male NU/NU mice from Charles RiverLaboratories International, Inc. (Wilmington, Mass.), about 8 week-old,were used here, and fed a white chow (AIN-93G Purified Diet, Harlan,Madison, Wis.) and water, and housed under standard conditions. TC1 wildtype mES cells were labeled with ¹¹¹In-MTTI-157 at the dye concentrationof 20 μM with about 50 μCi/ml per labeling mix according to the methoddescribed before. Then about 4-5 μCi of ¹¹¹In-MTTI-157 labeled cells in0.1 ml PBS (5×10⁶ per mouse) was injected into NU/NU mice by tail veinor delivered subcutaneously on the left shoulder. The mice were followedby SPECT/CT imaging (NanoSPECT/CT, Bioscan, Inc., Washington, D.C.) withscans at 1 h and 19 h for injection subcutaneously, and at 30 min and 18h for injection by tail vein.

The whole body radioactivity was measured through day 9 after injectionby placing the mouse in a dose calibrator (Capintec).

The SPECT image parameters were 1.0 mm/pixel, 256×256 frame size and 60sec per projection with 24 projections. Acquisition time wasapproximately 30 min. During imaging, the animal was anesthetized with1-2% isoflurane in 1.5 liters/min oxygen. A CT scan was acquired priorto the SPECT scan and was performed at standard resolution, using a 45kVp voltage and 500 milliseconds exposure time. The CT and SPECTreconstruction was performed using InVivoScope 1.43 software (Bioscan,Washington, D.C.).

Optical images (Pearl Imager, LiCor Biosciences, Lincoln, Nebr.) wereacquired on day 0 and at intervals through day 28. After imaging, thelungs, liver, spleen and teratoma were removed, imaged and then imbeddedin OCT in preparation for frozen section fluorescence microscopy. Slideswere counter stained with the general membrane stain D275 (Invitrogen,Em: 484 nm, Ex: 501 nm). Slides were viewed in an Olympus IX 70 InvertedLight Microscope (Olympus America, Inc., NY) using filters for FITC (forD275) and Cy5 (for MTTI-157). The frozen sections were also stained withhematoxylin-eosin (HE).

For the injection by tail vein, the SPECT/CT image (FIG. 3) showed thatthe radioactivity accumulated in the lungs at 1 h after injection andthen distributed to liver and spleen by 19 h. For the mouse injectedsubcutaneously on the left shoulder, the radioactivity was retained inthe shoulder and did not distribution to other tissue through 18 h (FIG.4). The whole body radioactivity measurements corrected for decay (FIG.5) indicated that the radioactivity was retained in the body through day9. Collectively, these data indicate that the ¹¹¹In-MTTI-157incorporated into the stem cells and remained cell bound—the activitywas not observed in organs of excretion (kidneys, bladder or intestinaltract)—and the activity remained in the body over 9 days—thus was stablein vivo. The time point in FIG. 5 at Day7 was lower than other pointsdue to the low radioactivity and measurement accuracy.

Optical images in mice injected with ¹¹¹In-MTTI-157 labeled stem cellssubcutaneously on the left shoulder was performed through day 14. Thestem cells in the shoulder developed into a solid teratoma over the 14days, measuring about 250×170 mm (1×w) when removed on day 14. Theoptical images (FIG. 6) show a strong fluorescence signal retained onthe shoulder injected with ¹¹¹In-MTTI-157 labeled stem cells though day9, while no fluorescence is observed on the opposite shoulder. Thefluorescence signal appears to grow weaker by day 13 and 14 as theteratoma grew larger, but this was due to the signal now associated withcells located within the more central mass and at depth, so detection byoptical imaging was more limited as expected. The optical images (FIG.7) of the teratoma removed from the mouse and cut in half shows thestrongest fluorescence signal in the teratoma center. The optical images(FIG. 7) of the excised liver, lungs and spleen from a mouse sacrificedon day 10 after injection by tail vein of ¹¹¹In-MTTI-157 labeled stemcells showed fluorescence signal from the tissues. Fluorescencemicroscopy of these same tissues also shows fluorescence in frozensections from liver, lungs, spleen and teratoma (FIG. 8). HE staining(FIG. 9) indicates that teratomas (red arrow showed) formed in the lungsand liver from the mouse injected by tail vein with ¹¹¹In-MTTI-157labeled stem cells and no teratoma was observed in the spleen.

Example 5. Stem Cells Labeled with MTTI-157 to Define Optimum DyeConcentration for High Dye Incorporation and Cell Labeling Efficiency:Analysis by Flow Cytometry

Wild type mouse embryonic stem (mES) cells BL/6 were cultured undernormal conditions, trypsinized, collected from the dishes, and washedtwice with high glucose DMEM without serum. Then the washed cells wereresuspended in Diluent C at the concentration of 2×10⁷ cells/ml.Immediately, prior to staining, a set of 2× working MTTI-157 stocksolutions at 4 μM, 20 μM, 40 μM were prepared in Diluent C. Cellsuspensions of 1 ml were added rapidly to the 1 ml of 2× working dyestock and immediately mixed by pipetting (final concentrations of 2 μM,10 μM, and 20 μM). After incubation for 5 min at RT with pipetting upand down constantly, the staining was stopped by adding an equal volumeof 1% BSA and incubating for 1 min at RT. Subsequently, the cellssuspension was centrifuged at 400 g for 10 min. Then the cells werewashed 3 times with 10 ml of DMEM with serum to remove unbound dye.After labeling, the cell viability was determined by trypan blue. Afterwashing, the cells were resuspended in 1% paraformaldehyde for flowcytometry analysis. Unlabeled BL/6 cells were used as a control.

Wild type mES cells BL/6 were labeled with MTTI-157 at the finalconcentrations of 2 μM, 10 μM, and 20 μM and analyzed by flow cytometry.Trypan blue staining indicated that the cell viability was over 90%after labeling at the dye concentrations from 2 μM to 20 μM. Flowcytometry analysis showed that the percent of labeled cells in totalcells was 9.8% at 2 μM, 28.7% at 10 μM, and 34% at 20 μM (FIG. 10). Theincorporation of MTTI-157 dye into stem cells is low, even at the highdye concentration of 20 μM. For further study, it is necessary toimprove the dye incorporation into the stem cells.

Example 6. BL/6 Wild Type Mouse Embryonic Stem Cells (mES) Labeled with¹¹¹In-MTTI-157 in Nude Mice: SPECT/CT and Optical Imaging: SecondExample

The fate of BL/6 wild type mES cells labeled with ¹¹¹In-MTTI-157delivered by tail vein or subcutaneously on the shoulder was followed bySPECT/CT and optical imaging.

BL/6 wild type mouse embryonic stem cells (mES) were labeled with¹¹¹In-MTTI-157 at the concentration of 12 μM and about 250 μCi/mlaccording to the method described before. The labeled cells (5 in 0.1 mlPBS (5×10⁶ per mouse) were injected into NU/NU mice by tail vein orsubcutaneously on the left shoulder. SPECT/CT imaging was done at 30min, 21 h and day 4 after injection subcutaneously, and at 1 h, 22 h andday 4 when injected by tail vein. Optical imaging was performed throughday 11 for the subcutaneous delivery of cells. The whole bodyradioactivity was measured through day 7 by placing the animal in a dosecalibrator.

The lungs, liver, and spleen were removed from the mouse injected bytail vein when sacrificed on day 19, and the teratoma was removed frommouse injected subcutaneously on the left shoulder when sacrificed onday 15. Excised organs and teratoma were imaged on the Li-CorBiosciences Pearl Imager, and then set in OCT for frozen sections. Thefrozen sections were stained with the general membrane fluorescent stainD275 (Invitrogen, Em: 484 nm, Ex: 501 nm) and detected on an Olympus IX70 Inverted Light Microscope (Olympus America, Inc., NY) using a FITCfilter for D275 and a Cy5 filter for MTTI-157. D275 is a cell membranestain and was used here to stain the field of view. The frozen sectionswere also stained with hematoxylin-eosin (H&E) for standard microscopy.

Following the delivery by tail vein, the SPECT/CT image (FIG. 11) showsradioactivity accumulated in the lungs by 1 h following injection whichthen distributed to the liver and spleen by 22 h. On day 4,radioactivity in the liver and spleen is still observed. Noradioactivity is seen in the kidneys, bladder, intestine or othertissues. The lack of radioactivity in these organs is an indication ofthe stability of ¹¹¹In-MTTI-157 in vivo once incorporated into the cellmembrane. For the mouse injected with cells subcutaneously on the leftshoulder, the SPECT/CT images showed that the radioactivity is retainedin the shoulder without distribution to other tissue and even to themuscle close to the injection site through day 4 (FIG. 12). The wholebody radioactivity measurements (FIG. 5) indicate that the radioactivityis unchanged from 2 h to 21 h after injection, but then decreasesslightly at 91 h and remain at this level through 165 h (7 days).

Optical images were taken of mice with ¹¹¹In-MTTI-157 stem cellsdelivered subcutaneously on the left shoulder through day 11. Thesecells grew into an obvious mass termed a teratoma that retained (FIG.13) a strong fluorescent signal though day 7. The signal was stillpresent but weakened on day 11 due to the growth of the teratoma. Theteratoma was removed after sacrifice and shown in FIG. 5 is the opticalimage of the mass cut in half to expose the inner area that has retainedthe strong fluorescent signal. The liver, lungs and spleen (FIG. 14)from the mouse sacrificed on day 19 after injection of ¹¹¹In-MTTI-157stem cells by tail vein shows different degrees of fluorescence signalin each of these tissues. The muscle was used as background for the autofluorescence. Fluorescence microscopy (FIG. 15) of these same tissuesshows obvious MTTI-157 fluorescence signal from the lungs and teratoma,the liver and spleen show limited but evidence of fluorescent areas. Thelungs and liver were viewed by H&E staining (FIG. 16) and the formationof teratomas (red arrows) in these organs is evident from the mouseinjected with ¹¹¹In-MTTI-157 stem cells by tail vein. No evidence of ateratoma was noted in the spleen.

Example 7. MTTI-157 Labeled LS174T Cells: Test for Improved LabelingEfficiency

For improved labeling efficiency, LS174T cells labeled with MTTI-157 wastested with different conditions as shown in Table 1 and Table 2. Themethod for LS174T cells labeling with MTTI-157 was as described asbefore. Briefly, LS174T cells were trypsinized, collected, and washedtwice with MEM without serum and then resuspended in Diluent C.Immediately, prior to staining; a 2× working dye stock solution inDiluent C was prepared. The cell suspension was added rapidly to anequal volume of the 2× working dye solution and immediately mixed bypipetting up. After incubation for 10 min at RT with pipetting up anddown constantly, the staining was stopped by adding 1% BSA andincubating for 1 min at RT. Subsequently, the cell suspension was spunat 400 g for 5 min. The cells were washed 3 times with 10 ml of MEM withserum to remove unbound dye. After labeling, the labeled cells werefixed in 2% paraformaldehyde and analyzed by flow cytometry.

Set 1:

MTTI-157 was dissolved in ethanol at the concentration of 2 μg/μL.LS174T cells were labeled with MTTI-157 with the conditions as shown inTable 1.

TABLE 1 Labeling conditions for LS174T cells with MTTI-157 dissolved inethanol; results of flow cytometry analysis Final MTTI-157 0 2 10 20 210 20 2 10 20 concentration (μM) Final cells 1 1 1 1 10 10 10 1 1 1concentration (×10⁷/ml) DMSO in 2x 0 0 0 0 0 0 0 2 2 2 working dye stock(%) % labeled cells in 4.8 12.6 62.7 74.3 29.5 57.9 75.0 23.7 55.9 71.1total cells by FACS

Set 2:

MTTI-157 was dissolved in DMSO at the concentration of 2 μg/μL. LS174Tcells were labeled with MTTI-157 at the condition as shown in Table 2.

TABLE 2 Labeling conditions for LS174T cells with MTTI-157 dissolved inDMSO; results of flow cytometry analysis Final dye 0 10 20 20 40 40 80concentration (μM) Final cell 10 10 10 20 10 20 10 concentration(×10⁷/ml) DMSO in 2x 0 2 4 4 8 8 16 working dye stock (%) % labeledcells in 0.0 97.3 97.9 98.6 99.2 98.5 99.5 total cells by FACS

For improved cell labeling with MTTI-157 the following were tested: twosolvents (ethanol and DMSO) to dissolve MTTI-157; different cellconcentrations; and different dye labeling concentrations.

In Set 1, MTTI-157 was dissolved in ethanol. The flow cytometry analysis(Table 1) showed that the dye concentration had an apparent effect onthe labeling efficiency, while no obvious effect on cell labelingefficiency with increase in cell concentration or the presence of DMSOin the labeling mixture. The labeling efficiency improved as the dyeconcentration increased. Even at the highest final dye concentration of20 μM, the labeling efficiency was about 70%. At the dye concentrationof 2 μM, the cell labeling efficiency was higher only if the cellconcentration was high, 10×10⁷/ml, or if DMSO was present in thelabeling mixture. But in either case the labeling efficiency did notapproach 70%. At the dye concentration of 10 μM and 20 μM, no obviousdifference was shown between cell concentration or the presence of DMSOin the labeling mixture.

In Set 2, MTTI-157 was dissolved in DMSO. The flow cytometry analysis(Table 2) showed that even at the final dye concentration of 10 μM, thelabeling efficiency was 97.3%, which in Set 1 was only about 60% whenMTTI-157 was dissolved in ethanol. This study indicates that DMSO may bebetter than ethanol as solvent for incorporating MTTI-157 into the cellmembrane.

Example 8. Fluorescent and ¹¹¹In Labeled Agent for Optical and SPECTCell Tracking

LS-174T cells were labeled with ¹¹¹In-MTTI-157. Fluorescence microscopywas used to evaluate incorporation and retention in cells thru 8 days.SPECT and optical imaging was used to track distribution and retentionin mice of ¹¹¹In-MTTI-157-labeled LS-174T cells (about 10 μCi) deliveredby tail vein.

¹¹¹In -MTTI-157 was incorporated into LS-174T cancer cells as a modelfor stem cells. A small volume stock solution (about 100 μL) of MTTI-1571-2 μg/μL in 100% ethanol was made fresh each time, with storage for nolonger than a week. The MTTI-157 stock solution was stored in a screwcapped Eppendorf tube in the dark at RT. The labeling reaction was with2-15 μL (61-414 μg) of MTTI-157 stock solution in a screw cappedEppendorf tube. The water content of the final radiolabeling reactionwas at or less than 10%. Too high a water content and a precipitate canform. For the labeling reaction to a volume of ¹¹¹InCl₃ (about 1 μL) wasadded an equal volume of 0.25 M ammonium acetate buffer pH 5.2. Thesample was left for 10-15 min before transfer to the dye solution. The¹¹¹In acetate volume transferred was about 1 depending on activityrequired and the aqueous volume needs to be considered to avoid theprecipitate. The transfer was quick with repeated mixing with thepipette. If the volume is ample, a quick spin on the vortex is done forthorough mixing. The labeling reaction mixture was placed in a waterbath pre-set to 40° C. After about 5 min the water bath was turned off,and the sample left for 1-2 hrs. If there is significantevaporation-refluxing-after the incubation, the tube is spun to bringdown all liquid before analyzing.

Labeling efficiency was determined by C8 HPLC (Phenomenex, Luna 5 u, C8(2), 100 A, 250×4.6 mm, 5 micron) flow rate of 1 mL/min with a mobilephase A (90% methanol, 0.1% TFA) and mobile phase B (100% methanol, 0.1%TFA). Initial conditions were 95% A for 1 minute then gradient to 10% Aat 15 minutes, and 5% at 20 min, then back to 95% at 30 min. ITLC waswith silica strips (Gelman) with solvent of methanol, and 1% TFA.

The ¹¹¹In -MTTI-157 incorporated into cells was followed in culture todetermine the fate of both labels (fluorescence and ¹¹¹In), ability topass on to subsequent generations, and cell's retention ofviability/proliferation by following cells in culture. The ¹¹¹In-MTTI-157 was incorporated into LS-174T cells according to the methodsdescribed before for the CellVue family of dyes. Briefly, LS174T cellsat 90% confluency were trypsinized, and washed twice with MEM withoutserum. The cells were resuspended in Diluent C with 2×10⁷ cells/ml.Immediately, prior to staining, a 2-fold concentrated working dye stocksolution was prepared in Diluent C at dye concentrations of 4 μM, 20 μMand 40 μM and with 1.7-5.2 ¹¹¹In for in vitro studies and about 500 μCifor in vivo studies. To 100 μL of a cell suspension was added rapidly100 μL of a 2× working dye solution with immediate mixing by pipettethat was continued for 10 min at RT. The dye incorporation wasterminated by adding an equal volume (200 μL) of 1% BSA and leaving for1 min at room temperature (RT) before the cells were spun down (400×gfor 10 min) and washed 3 times with 10 ml of MEM with serum to removeany unincorporated dye. After washing, cell viability was determined bytrypan blue.

The ¹¹¹In-MTTI-157 labeled LS-174Tcells were seeded into 12-well plateswith about 10⁶ cells per well to grow under normal culture conditions toevaluate cell viability, dye stability, and dye partitioning to daughtercells over time. The cell-associated fluorescence was detected from theplates on an Olympus IX 70. Inverted Light Microscope (Olympus America,Inc., NY) using a Cy5 filter on days 0, 3 and 8 after seeding. On eachday, samples of cells were collected, counted for cell number with ahemocytometer and radioactivity per 10⁶ cells determined by gamma wellcounter Na (TI) (Cobra II Auto-Gamma, Packard Instrument Co, DownerGrove, Ill.).

¹¹¹In-MTTI-157 labeled LS174T cells were administered to mice to followtheir fate and that of the labels they carried. All animal experimentswere approved by the Institutional Animal Care and Use Committee of theuniversity. Male SKH-1 mice about 8 week-old (Charles RiverLaboratories, Inc., Wilmington, Mass.) were housed under standardconditions and fed white chow (AIN-93G Purified Diet, Harlan, Madison,Wis.). LS174T cells were prepared labeled with ¹¹¹In-MTTI-157 as above,and about 5×10⁶ cells with 10-12 μCi was injected by tail vein permouse.

The mice were followed by SPECT/CT imaging (NanoSPECT/CT, Bioscan, Inc.,Washington, D.C.) with scans at 30 min, 1 h, 24 h, 72 h, 120 h and 168 hfollowing injection. The SPECT image parameters were 1.0 mm/pixel,256×256 frame size and 60 sec per projection with 24 projections.Acquisition time was approximately 30 min. During imaging, the animalwas anesthetized with 1-2% isoflurane in 1.5 L/min oxygen. A CT scan wasacquired prior to the SPECT scan and was performed at standardresolution, using a 45 kVp voltage and 500 milliseconds exposure time.The CT and SPECT reconstruction was performed using InVivoScope 1.43software (Bioscan, Washington, D.C.).

Optical images (Pearl Imager, LiCor Biosciences, Lincoln, Nebr.) wereacquired on day 0 and at intervals through day 28. To facilitatedetection of signal at depth, such as in the lungs, some animals werekilled, the chest and abdomen opened to expose internal organs, and thenimaged. Untreated mice served as control. After imaging, the lungs,liver and spleen were removed, imaged and then imbedded in OCT inpreparation for frozen section fluorescence microscopy. Slides werecounter stained with the general membrane stain D275 (Invitrogen).Slides were viewed in an Olympus IX 70 Inverted Light Microscope(Olympus America, Inc., NY) using filters for FITC (for D275) and Cy5(for MTTI-157).

The MTTI-157 at 2 μg/μL ethanol was labeled with ¹¹¹In in 0.25 Mammonium acetate, pH 5.2. After 1 h greater than 90% radiochemicalpurity was attained as shown by C18 HPLC in FIG. 17. The labeled productelutes with retention time of 18.7 min. Specific activity of 10 μCi/μgwas reproducibly attained, and a maximum specific activity of 74 μCi/μgwas achieved showing 89% radiochemical purity.

To determine the fate of both ¹¹¹In and fluorescence labels in vitro,cells were labeled with ¹¹¹In-MTTI-157 using final sample concentrationsof 2 μM, 10 μM and 20 μM and grown under normal culture conditions for 8days. The stability of both labels was determined by fluorescencemicroscopy and nuclear counting. After labeled dye incorporation trypanblue staining showed greater than 95% cell viability (data not shown).When a 40 μM sample concentration was used cell viability dropped toabout 90% (data not shown). Bright light and corresponding fluorescencemicroscopy of cells labeled with 20 ¹¹¹In-MTTI-157 are shown in FIG. 18from days 0, 3 and 8. Cells show normal growth over the 8 days anddemonstrate that with proliferation succeeding generations retained thefluorescence marker with no apparent effect on cell viability. With theincrease in labeled dye concentration, fluorescence intensity increased.

The cell-associated radioactivity with 2, 10 and 20 μM ¹¹¹In-MTTI-157 ondays 0, 3, and 8 is shown in FIG. 19a . The loss of radioactivity fromday 0 to day 3 is likely due to the loss of cells from initial platting,once the cells were established as indicated on days 3-8, the cellassociated radioactivity did not change at each dye concentration.However, the cell number showed an increase at each dye concentrationfrom day 3 to day 8 and the trend in cell number (growth) was comparableto the control cells with no dye (FIG. 19b ).

¹¹¹In-MTTI-157 labeled LS174T cells were injected into SKH-1 mice bytail vein to evaluate the fate of both labels in vivo. Mice werefollowed by SPECT/CT over the course of 7 days and by optical imagingover 28 days. SPECT/CT are shown in FIGS. 20a and 20b , in the earliestscan at 30 min and 1 h radioactivity is restricted to the lungs and thenseen to redistribute within a day to the liver and spleen with someremaining in the lungs in the case of image FIG. 20 b.

SPECT scans through day 7 show the distribution unchanged with onlylungs, liver and spleen apparent out to day 5-7. No radioactivity inkidneys and bladder was obvious on any day. The lack of activity in gut,kidneys and bladder indicates the stability of the labeled dye on thecells.

Whole body radioactivity measurements by dose calibrator is shown in(FIG. 21) as the percent of injected activity remaining (half-lifecorrected) starting at 5 min (on day 0) through day 14. The loss ofactivity from day 0 to day 10 (across four half-lives) was about 23%.Beyond 10 days radioactivity was too near background for accuratedetermination. These data show that ¹¹¹In-MTTI-157 labeled cells werestable in vivo, and the labeled dye ¹¹¹In-MTTI-157 itself is stable.

Whole body optical images acquired of the same mice but followed throughday 28 are shown in FIG. 22. As with SPECT the early optical images showfluorescence signal in the lungs on day 0 that is still apparent on day28 (in comparison to signal in control animal lungs). As with the SPECTscans by the first day the signal distributes to the liver and spleen,and is still obvious in the optical scans through day 28. Confirmationwas obtained from excised organs. Shown are excised lungs, liver andspleen from an untreated mouse (top row) and treated mice optical scansfrom days 1, 7, 14 and 28 of excised lungs, liver and spleen (FIG. 23).

Further examination of lungs, liver and spleen by fluorescencemicroscopy confirm the retention of the MTTI-157 in these tissuesthrough day 28 as shown in FIG. 24. The sections were treated with themembrane lipophilic dye D275 (green) to define the field of view and theMTTI-157 signal (red) is apparent in lungs through day 14 and in liverthrough day 28. These data are consistent with the results from SPECT/CTand optical imaging.

SPECT/CT scans of mice receiving ¹¹¹In-MTTI-157 labeled cells by tailvein showed radioactivity restricted to the lungs in the earliest scanson Day 0, but redistributed by the next day to include the liver andspleen. Through Day 7 distribution was unchanged with only lungs, liverand spleen apparent. No indication of ¹¹¹In instability was observed asevident by the absence of activity in gut, kidneys and bladder on anyday. Parallel whole body activity measurements (decay corrected)confirmed ¹¹¹In retention out thru Day 14; beyond that time activity wastoo near background for accurate determination.

Corresponding whole body optical images showed fluorescence in the lungsand liver out through Day 28 that was confirmed by optical scans of theexcised organs, including spleen. Fluorescence microscopy of tissuefrozen sections confirmed cell associated MTTI-157 signal in lung thruDay 21 and liver and spleen thru Day 28.

This dual labeled cell marker, ¹¹¹In-MTTI-157, has provided a means tomonitor the in vivo fate of cells out through 28 days in vivo with noevidence of label instability. This cell marker will be evaluatedfurther for tracking the fate of stem cells in vivo for potential use incell-based therapies.

Example 9. Dyes Transfer Flow Cytometry Analysis

The human colon cancer cell line LS174T was used here as a model toidentify the optimal dyes marker with minimal transfer to neighbor cellsfor cell tracking. A family of DiD dyes (Ex: 648 nm, Em: 667 nm) withvariable lipophilic aliphatic tails (Cm/Cn here refer to lengthslipophilic aliphatic tails in the number of C atoms) were investigated:MT279 (C22/C3), MT280 (C22/C12), MT284 (C14/C3), MT285 (C14/C14), CN1012(C20/C20), MTTI-157 without the DOTA (C22/C22). LS174T cells werecultured in minimal essential medium (MEM) with nonessential aminoacids, 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at37° C. in a humidified atmosphere with 5% CO₂ and were passaged weekly.

Optimization of Dye Concentrations

Cell labeling efficiency was observed as being different with the sixdyes, so did this study to select a concentration that would give atleast 100% of cells labeled with a fluorescence intensity that wassimilar among the group of six dyes. Not all dyes labeled cells at asimilar level. Some dyes (like MT284) gave results that were noted asbeing too bright for reliable FACS analysis. Thus different dyeconcentrations were investigated looking for the ideal concentration foreach dye.

Four working dye stock solutions were investigated. LS174T cells weretrypsinized, collected, and washed twice with MEM without serum when theflask was 90% confluent. The washed cells were resuspended in Diluent Cat the concentration of 2×10⁷ cells/ml. Immediately, prior to staining,2× working dye stock solutions (1 μM, 4 μM, 10 μM or 20 μM) in Diluent Cwere prepared. Then 500 μl of cell suspension was added rapidly into 500μl of 2× working dye and immediately mixed by pipetting. Afterincubation for 10 min at room temperature with pipetting up and downconstantly, the staining was stopped by adding equal volume (1000 μL) of1% BSA and incubating for 1 min at room temperature. Subsequently, thecells suspension was centrifuged at 400 g for 10 min. Then the cellswere washed 3 times with 10 ml of MEM with serum to remove unbound dye.After labeling, the cell viability was determined by trypan blue. Thelabeled cells were fixed in 2% paraformaldehyde and analyzed by flowcytometry.

The cells were labeled at 0.5 μM, 2 μM, 5 μM or 10 μM and analyzed byflow cytometry to determine the optimum concentration. The flowcytometry analysis (FIG. 25) showed that the fluorescence intensity percell increased as the dye concentration increased. At the sameconcentration, the fluorescence intensity per cell for the dyesindicated that MT284 (C14/C3)>MT279 (C22/C3)>MT280 (C22/C12)>MT285(C14/C14)>CN1012 (C20/C20)>MTTI-157 (C22/C22). Especially for MT284, thefluorescence intensity was over the detection range of flow cytometryeven at low labeling concentration (0.5 μM). The results suggest that itis easier for dyes with shorter carbon tails to incorporate into thecell membrane. For dyes MT279, MT280, MT284 and MT285, 100% of the cellswere labeled even at the lowest dye concentration of 0.5 μM, and ofcourse remained at 100% as dye concentration increased. While for dyeCN1012, the percent of cells labeled at the concentrations of 0.5 μM, 2μM, 5 μM or 10 μM was 93.1%, 99.5%, 99.9%, 100% respectively, and forMTTI-157 it was 71.5%, 92.3%, 98.6%, 99.4%, respectively (FIG. 26).

Evaluation of Dye Transfer: Co-Culture of Dye Labeled and UnlabeledCells

LS174T cells were trypsinized, collected, and washed twice with MEMwithout serum and labeled with the six dyes according to the methoddescribed above. Briefly, the washed cells were resuspended in Diluent Cat the concentration of 2×10⁷ cells/ml. immediately, prior to staining,2× working dye stock in Diluent C were prepared. Study sets aredescribed below. One (1) ml of the cell suspension was added rapidly to1 ml of 2× working dye solution and immediately mixed by pipetting.After incubation for 10 min at room temperature with pipetting up anddown constantly, the staining was stopped by adding an equal volume (2ml) of 1% BSA and incubating for 1 min at room temperature.Subsequently, the cells suspension was spun at 400 g for 10 min. Thenthe cells were washed 3 times with 10 ml of MEM with serum to removeunbound dye. The cell viability was determined by trypan blue staining.The dye labeled cells were co-cultured with an equal number of unlabeledcells under normal culture conditions. Samples were removed at 0 h, 24 hand 48 h after initiation of co-culture and fixed in 2% paraformaldehydefor flow cytometry analysis.

Set 1:

Six dyes: MT279, MT280, MT284, MT285, CN1012, MTTI-157, were used forcell labeling at the final concentration in the labeling mix of 2 μM foreach dye.

LS174T cells were labeled with the 6 dyes at 2 μM and co-cultured withan equal number of unlabeled cells. FIG. 27 shows the percent of labeledcells at different time points after co-culture from flow cytometryanalysis. The percent of unlabeled cells incorporating dye from labeledcells for each dye at 24 h, 48 h after co-culture compared to that at 0h is shown in Table 3. From the data in Table 3 it appears that MT284 isthe best dye, showing no transfer to unlabeled cells. But, in FIG. 27,dye MT284 showed at 0 h of co-culture, already about 100% of the cellswere labeled. It is possible that the dye transfer occurred when thesamples were fixed and stored at 4° C. and held for 48 h prior to flowcytometry analysis. So these data may not accurately represent the truesituation. Therefore to avoid this possibility, in the next study forthe 0 h sample, the labeled cells and unlabeled cells were collected andfixed separately, and then mixed immediately before flow cytometryanalysis.

TABLE 3 Percent of unlabeled cells incorporating dye from labeled cellsfor each dye at 24 h and 48 h of co-culture compared to the value at 0h. MT279 MT280 MT284 MT285 CN1012 MTTI-157 24 h 16.68 9.03 −0.74 4.2320.69 22.39 48 h 18.36 13.83 −2.06 6.8 22.6 22.74

Set 2:

Six dyes: MT279, MT280, MT284, MT285, CN1012, MTTI-157 were used for thecell labeling at the optimum concentration determined. The finalconcentration in the labeling reaction for the dyes were respectively:0.5 μM, 2 μM, 0.25 μM, 2 μM, 5 μM and 10 μM. Based on the results ofoptimization of dye concentration study, at these concentrations thecells were 100% labeled (all cells carried a label) and had a similarmean fluorescence intensity when analyzed by FACS.

Based on the results of the optimization of dye concentration study, thelabeling concentrations for the dyes were selected as follows: 0.5 μM, 2μM, 0.25 μM, 2 μM, 5 μM and 10 μM for dyes MT279, MT280, MT284, MT285,CN1012, and MTTI-157 respectively. These concentrations were used toensure that 100% of the cells were labeled and carried a similar meanfluorescence intensity. Also, for the sample at 0 h, the labeled cellsand unlabeled cells were collected and fixed separately, and mixedimmediately before flow cytometry analysis. The flow cytometry resultsare shown in FIG. 28. The percent of unlabeled cells incorporating dyefrom labeled cells for each dye at 24 h and 48 h of co-culture comparedto that at 0 h is shown in Table 4. The results collectively show thatat low dye concentrations, i.e., 0.5 μM, 2 μM, and 2 μM for MT279,MT280, and MT285, no dye transfer to unlabeled cells occurred. Dye MT284appears to be the most efficient dye to transfer. Dye MTTI-157 showedtransfer as well but with 22% at 24 h and 28% at 48 h. Dye CN1012 showedlower transfer than MTTI-157, with 8% at 24 h and 15% at 48 h.

TABLE 4 Percent of unlabeled cells incorporating dye from labeled cellsfor each dye at 24 h and 48 h after co-culture compared to the value at0 h. MT279 MT280 MT284 MT285 CN1012 MTTI-157 24 h −6.13 −3.59 60.33 −5.38.115 22.17 48 h −2.9 −0.1 58.64 4.92 14.93 28.45

Set 3:

Since in Set 2, some dyes (MT279, MT280 and MT285) showed no transfer atthe concentrations selected for testing, Set 3 was done at another setof concentrations to verify whether dye will transfer if a higherconcentration was used, and it did. Six dyes: MT279, MT280, MT284,MT285, CN1012, and MTTI-157 were used for the cells labeling. The finalconcentrations in the labeling reaction for the dyes were 5 μM exceptfor MT284, which was tested again at 0.25 μM.

LS174T cells were labeled with the six dyes MT279, MT280, MT284, MT285,CN1012, and MTTI-157, respectively. The final dye concentration in thelabeling reaction in this case was 5 μM except for MT284, which wastested again at 0.25 μM.

The labeled cells were co-cultured with unlabeled cells, and as beforeat 0 h, the labeled cells and unlabeled cells were collected and fixedseparately, and mixed immediately before flow cytometry analysis. Theflow cytometry results are shown in FIG. 29. The percent of unlabeledcells incorporating dye from labeled cells for each dye at 24 h and 48 hof co-culture compared to that at 0 h is indicated in Table 5. Thisstudy showed that at this higher dye concentration (5 μM), all six dyestransferred from labeled cells to unlabeled cells. As indicated in Table5, MT285 showed the least transfer, and again PITR284 showed the highesttransfer even at the lower concentration (0.25) (about all the unlabeledcells took in dye from labeled cells).

TABLE 5 Percent of unlabeled cells incorporating dye from labeled cellsfor each dye at 24 h and 48 h of co-culture compared to the value at 0h. MT279 MT280 MT284 MT285 CN1012 MTTI-157 24 h 25.88 10.76 48.23 −2.3612.9 6.93 48 h 39.42 26.85 48.36 17.27 24.46 22.16

Set 4:

Based on Set 2 and Set 3 results, Set 4 was done. Here only these threedyes: MT280, MT285, and MTTI-157 were used for the cells labeling. Thesethree dyes were the best and each was tested at 3 concentrations todetermine the best concentration for labeling. The dye finalconcentrations for labeling were 0.5 μM, 2 μM, and 5 μM.

LS174T cells were labeled with the six dyes separately and co-culturedwith unlabeled cells to identify the optimal dye marker showing minimaltransfer to neighboring cells. Samples were collected at 0 h, 24 h and48 h of co-culture and fixed in 2% paraformaldehyde for flow cytometryanalysis.

From the previous studies, MT280 and MT285 showed the least amount ofdye transfer and were selected for further evaluation along withMTTI-157. In this study, the three dyes were each labeled at thefollowing three concentrations: 0.5 μM, 2 μM, and 5 μM. (BUT these threeconcentrations are only ideal for MT280 and MT285, and not ideal forMTTI-157, which does not label cells as well at these lowconcentrations). As before, the labeled cells were co-cultured withunlabeled cells and samples were collected at 0 h, 24 h, and 48 h ofco-culture and analyzed by flow cytometry. For the sample at 0 h, thelabeled cells and unlabeled cells were collected and fixed separately,and mixed immediately before flow cytometry analysis. The flow cytometryresults are shown in FIG. 30 as the percent of cells labeled. As isobvious in FIG. 30 for MTTI-157 the percent of labeled cells is low atthese dye concentrations, especially at 0.5 μM. The more ideal labelingconcentration for MTTI-157 is 10-20 μM. The percent of unlabeled cellsincorporating dye from labeled cells for each dye at 24 h and 48 h ofco-culture compared to that at 0 h is shown in Table 6. These data showthat for MT280 and MT285, almost no transfer occurred at the lowconcentration of 0.5 μM. For MTTI-157, at the concentration of 0.5 dyetransfer took place. At the concentrations of 2 μM and 5 all the threedyes transferred from labeled cells to unlabeled cells. At these two dyeconcentrations transfer to unlabeled cells with each of the three dyeswas similar as is apparent from the values in Table 6.

TABLE 6 Percent of unlabeled cells incorporating dye from labeled cellsfor each dye at 24 h and 48 h of co-culture compared to that at 0 h. 24h 48 h MT280 (0.5 μM) 1.675 1.29 MT280 (2 μM) 25.095 28.19 MT280 (5 μM)40.84 40.905 MT285 (0.5 μM) 5.07 7.835 MT285 (2 μM) 24.99 28.285 MT285(5 μM) 35.155 34.195 MTTI-157 (0.5 μM) 13.58 14.675 MTTI-157 (2 μM)23.555 25.615

In this specification and the appended claims, the singular forms “a,”“an,” and “the” include plural reference, unless the context clearlydictates otherwise.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Although any methods and materials similar or equivalent tothose described herein can also be used in the practice or testing ofthe present disclosure, the preferred methods and materials are nowdescribed. Methods recited herein may be carried out in any order thatis logically possible, in addition to a particular order disclosed.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made in this disclosure. All such documents arehereby incorporated herein by reference in their entirety for allpurposes. Any material, or portion thereof, that is said to beincorporated by reference herein, but which conflicts with existingdefinitions, statements, or other disclosure material explicitly setforth herein is only incorporated to the extent that no conflict arisesbetween that incorporated material and the present disclosure material.In the event of a conflict, the conflict is to be resolved in favor ofthe present disclosure as the preferred disclosure.

EQUIVALENTS

The representative examples are intended to help illustrate theinvention, and are not intended to, nor should they be construed to,limit the scope of the invention. Indeed, various modifications of theinvention and many further embodiments thereof, in addition to thoseshown and described herein, will become apparent to those skilled in theart from the full contents of this document, including the examples andthe references to the scientific and patent literature included herein.The examples contain important additional information, exemplificationand guidance that can be adapted to the practice of this invention inits various embodiments and equivalents thereof.

What is claimed is: 1-20. (canceled)
 21. A method for imaging, trackingand/or analyzing cells, comprising: labeling a sample of cells with acompound having the formula:

wherein B is a chelating moiety capable of complexing to a radioactivemetal; R and R₁ are substituent groups independently selected from thegroup consisting of alkyl, alkenyl, alkynyl, alkaryl and aralkyl, eachof R and R₁ comprising one or more linear or branched hydrocarbon chainshaving from about 2 to about 30 carbon atoms, and each of R and R₁ beingunsubstituted or substituted with one or more non-polar functionalgroups; L comprises a fluorophore with emission in the range from about650 nm to about 850 nm; R₂ is a spacer moiety having the formula:—(R₃)_(p)-Q-(R₄-Q′)_(q)-(R₅-Q″)_(r)-(R₆-Q′″)_(s)-(R₇Q″″)_(t), wherein R₃is an aliphatic hydrocarbon, R₄, R₅, R₆ and R₇ are independentlyselected from the group consisting of aliphatic, alicyclic or aromatichydrocarbons, heterocycles or CH₂C(CO₂H)═CH, Q, Q′, Q″, Q′″ and Q″″ arelinking groups independently selected from the group consisting ofamide, thiourea, hydrazone, acylhydrazone, ketal, acetal, orthoester,ester, anhydride, disulfide, urea, carbamate, imine, amine, ether,carbonate, thioether, sulfonamide, carbonyl, amidine and triazine, avalence bond, the aliphatic or alicyclic hydrocarbons having from about1 to about 12 linear carbon atoms, and the aromatic hydrocarbons havingfrom about 6 to about 12 carbon atoms; p, q, r, s and t each is 0 or 1;and n is 0 or 1; and imaging the cells via two or more modalitiescomprising optical imaging and radiological imaging.
 22. The method ofclaim 21, wherein the cells are stem cells.